Na.sup.+ /H.sup.+ antiporters are ubiquitous in living cells and have been assigned a large variety of important functions (Boron, W. F., & Boulpaep, E. L. (1983) J. Gen. Physiol. 81, 29-52; Krulwich, T. A. (1983) Biochim. Biophys. Acta 726, 245-264; Aronson, P. S. (1985) Annu. Rev. Physiol. 47, 545-560; Grinstein, S., ed. (1988) Na.sup.+/H.sup.+ Exchange CRC Press, Boca Raton, Fla.), the most straight-forward being the regulation of the cytoplasmic level of Na.sup.+. The antiporters are integral membrane proteins that carry out either electroneutral or electrogenic exchange of Na.sup.+ for H.sup.+ that is driven by primary ion translocation events. Several genes encoding eukaryotic (Sardet, C. Franchi, A., & Pouyssegur, J. (1989) Cell 56, 271-280; Tse, C. M., Ma, A. I., Yang, V. W., Watson, A. J., Levine, S., Montrose, M. H., Potter, J., Sardet, C., Pouyssegur, J., & Donowitz, M. (1991) EMBO J. 10, 1957-1967); Hildebrandt, F., Pizzonia, J. H., Reilly, R. F., Reboucas, N. A., Sardet, C., Pouyssegur, J., Slayman, C. W., Aronson, P. S., & Igarashi, P. (1991) Biochim. Biophys. Acta 1129, 105-108; Reilly, R. F., Hildebrandt, R., Biemesderfer, D., Sardet, C., Pouyessegur, J., Aronson, P. S., Slayman, C. W., & Igarashi, P. (1991) Am. J. Physiol. 261, F1088-94; Orlowski, J., Kandasamy, R. A., & Shull, G. E. (1992) J. Biol. Chem. 267, 9331-9339; Jia, Z. -P., McCullough, N., Martel, R., Hemmingsen, S., & Young, P. G. (1992) EMBO J. 11, 1631-1640) and prokaryotic (Goldberg, E. B., Arbel, T. Chen, J. Karpel, R., Mackie, G. A., Schuldiner, S., & Padan, E. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2615-2619; Karpel, R., Olami, Y., Taglicht, D., Schuldiner, S., & Padan, E. (1988) J. Biol. Chem. 263, 10408-10414; Pinner, E., Padan, E., & Schuldiner, S. (1992) J. Biol. Chem. 267, 11064-11068; Waser, M., Hess-Bienz, D., Davies, K., & Solioz, M. (1992) J. Biol. Chem. 267, 5396-5400) Na.sup.+ /H.sup.+ antiporters have been cloned, and the gene product of one of these genes, the nhaA gene from Escherichia coli, has been purified and studied in proteoliposomes (Taglicht, D., Padan, E., & Schuldiner, S. (1991) J. Biol. Chem. 266, 11289-11294). As with other secondary ion porters, no genetic or biochemical evidence has emerged for additional components of these transport systems.
In bacteria, electrogenic Na.sup.+ /H.sup.+ antiporters may be part of the mechanism for cytoplasmic pH homeostasis in the alkaline range of pH for growth (Krulwich, T. A. (1983) Biochim. Biophys. Acta 726, 245-264; Booth, I. R. (1985) Microbiol. Rev. 49, 359-378). In particular, electrogenic Na.sup.+ /H.sup.+ antiport activity appears to be required in order for extremely alkaliphilic bacteria, such as Bacillus firmus and Bacillus alcalophilus, growing at pH 10.5 to maintain a cytoplasmic pH of 8.3 (Krulwich, T. A., & Guffanti, A. A. (1989) Annu. Rev. Microbiol. 43, 435-463). We have taken advantage of Na.sup.+ /H.sup.+ antiporter-deficient mutants of Escherichia coli that have been constructed during the extensive recent dissection of the molecular biology and genetics of the antiport complement in E. coli by Padan, Schuldiner and their colleagues (Goldberg, E. B., Arbel, T. Chen, J. Karpel, R., Mackie, G. A., Schuldiner, S., & Padan, E. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2615-2619; Karpel, R., Olami, Y., Taglicht, D., Schuldiner, S., & Padan, E. (1988) J. Biol. Chem. 263, 10408-10414; Pinner, E., Padan, E., & Schuldiner, S. (1992) J. Biol. Chem. 267, 11064-11068). Using heterologous complementation of Na.sup.+ /H.sup.+ antiporter-deficient strains by cloned genes from alkaliphilic Bacillus firmus OF4, we have isolated and characterized one putative structural gene for a Na.sup.+ /H.sup.+ antiporter from the alkaliphile, designated nhaC (Ivey, D. M., Guffanti, A. A., Bossewitch, J. S., Padan, E., & Krulwich, T. A. (1991) J. Biol. Chem. 266, 23483-23489). It encodes a membrane protein whose deduced amino acid sequence is consistent with at least ten transmembrane regions and which markedly enhances the Na.sup.+ /H.sup.+ antiport activity of membranes from E. coli NM81; this mutant strain carries a deletion in one of the E. coli antiporter genes (nhaA). Another alkaliphile gene that restored partial Na.sup.+ resistance to E. coli NM81 was the product of the alkaliphile cadC gene, a probable cadmium binding protein (Ivey, D. M., Guffanti, A. A., Shen, A., Kudyan, N., & Krulwich, T. A. (1992) J. Bacteriol., in press).